Tularemia is a zoonotic disease caused by the highly virulent, facultative intracellular bacterium Francisella tularensis. Because of the virulence of this bacterium and its ease of aerosol transmission, it has been classified as a Category A bioterrorism agent. A live, attenuated tularemia vaccine containing F. tularensis LVS (live vaccine strain) is currently available for human use as an investigational new drug (IND). It is given by scarification; this delivery method is sub-optimal for rapid, mass vaccination and the product has reactogenicity issues. Therefore, an improved vaccine against tularemia is needed. The objective of this research is to develop an effective and easily administrated tularemia vaccine using four protein antigens: Tul4, FopA, Cpn60 (heat shock protein), and FT23KDAP (the 23 kDa protein) from F. tularensis as vaccine components, delivered with replication-defective adenovirus by the intranasal and transcutaneous immunization routes. These non-invasive vaccine delivery methods will undoubtedly enhance the compliance of a vaccination program. In this project, adenovirus and plasmid expression vectors encoding Tul4, FopA, Cpn60, and FT23KDAP will be constructed. In order to obtain an optimal vaccination protocol, immunization regimens with different combinations of adenovirus and plasmid vectors and with different sequences of intranasal, transcutaneous, and intramuscular delivery modes will be studied in a mouse model. The specific aims of this project are: Specific Aim #1: To develop a replication-defective, adenovirus-vectored vaccine against F. tularensis. Specific Aim #2: To compare the systemic and mucosal immunity elicited by the adenovirus-vectored vaccine delivered by combinations of intranasal, transcutaneous, and intramuscular administrations with that elicited by F. tularensis LVS through intradermal injection. Since no previous research has shown that genetic immunization with adenovirus or plasmid vectors could elicit protective immunity to F. tularensis, the proposed project is exploratory and developmental in nature.